You are on page 1of 42

12

Qualification of Water and Air


Handling Systems

Kunio Kawamura
Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan

I. PURPOSE OF VALIDATION

High-quality water and air are essential for the manufacture of pharmaceuticals.
Water is the most commonly used raw material in pharmaceutical manufactur-
ing; it is indirectly used in the manufacture of all dosage forms for cleaning
manufacturing equipment, and is also used as a major component which consti-
tutes injectable products. It is the one raw material that is usually processed by
the pharmaceutical manufacturer prior to use because it cannot be used as sup-
plied by the vendor. Water should be regarded as one of major raw materials for
the manufacture of pharmaceuticals whether or not it remains as a component of
the finished dosage form or is eliminated during the manufacturing process.
Water is thus an important raw material in GMP and in validating the manufac-
turing process.
Air supplied to the pharmaceutical manufacturing area or the air in the
environment of the pharmaceutical manufacturing area always contacts with
pharmaceuticals, and the quality of air influences the quality of the pharmaceuti-
cals manufactured, particularly in their cleanliness, particulates, and microbial
quality. Temperature and humidity in the manufacturing environment also influ-
ence the quality of the pharmaceuticals manufactured.
The importance of air quality and air handling system are described in
CFR 211-46 as part of GMP.
The USP identifies several grades of water that are acceptable for use in
pharmaceuticals, and also defines the quality of the environment or the quality
of air for the manufacturing of pharmaceuticals according to its criticality.

Copyright © 2003 Marcel Dekker, Inc.


Water and the environment must be periodically monitored for these qual-
ity attributes, and in some instances the results are not available for days after
the sample is obtained. Meanwhile, the water would have been used to manufac-
ture a great number of pharmaceutical products or else the products would have
already been exposed to the environment. Water treatment and air handling
systems are highly dynamic, and careful attention has to be paid to their opera-
tion, even though this may sometimes be somewhat unreliable. Consequently,
they must be validated and then closely monitored and controlled.
Validation is defined as “a documented program that provides a high de-
gree of assurance that a specific process, method, or system will consistently
produce a result meeting pre-determined acceptance criteria” [1].
The purpose of validation is to demonstrate the capability of the water
treatment and air handling system to continuously supply the required quantity
of water and air with the specified quality attributes. “Documented” means to
provide documented “evidence.” Validation provides the system owner with the
means of assessing when a water treatment and/or air handling system is operat-
ing outside established control parameter limits and provides a means for bring-
ing the system back into a state of control. It results in written operating and
maintenance procedures for personnel to follow, which in turn helps ensure
consistent system performance.

II. VALIDATION STRATEGY


A. Validation Concept
The basic strategy is to prove the performance of processes or systems under
all conditions expected to be encountered during future operations. To prove the
performance, one must demonstrate (document) that the processes or systems
consistently produce the specified quantity and quality of water and/or air when
operated and maintained according to specific written operating and mainte-
nance procedures. In other words, validation involves proving
1. Engineering design
2. Operating procedures and acceptable ranges for control parameters
3. Maintenance procedures
To accomplish this, the system must be carefully designed, installed, and tested
during and after construction, and therefore for a prolonged period of time under
all operating conditions.
Variations in daily, weekly, and annual system usage patterns must be
validated. For example, water may be drawn from the system for manufacturing
use only during normal working hours; there may be no demands on the system
at other times during the 24-hr cycle. The system may be idle on weekends and

Copyright © 2003 Marcel Dekker, Inc.


on holidays, which could extend for as long as 4 days or more. In addition,
many firms have annual plant maintenance shutdowns, typically in the summer,
and systems must be sanitized and restarted prior to use, and of course emer-
gency shutdowns can occur at any time and the system must be brought back
online. Systems with ion exchange resins (deionizers) must be at least partially
shut down to regenerate the resins when the chemical quality of the treated
water drops below a specified level. (This could be a matter of a few days or
even a few months, depending on the quantity of water processed through the
system and other factors.) For the air handling system, the same kinds of issues
exist. Clean rooms should be maintained at their required cleanliness level, even
during the time of no manufacturing operation. If the cleanliness is broken or
the air handling system stops, the whole clean area has to be made clean accord-
ing to the initial validation procedure and assessment. Water treatment and/or
air handling systems must be validated under all of these normal operating con-
ditions in order to prove the adequacy of the engineering design and the effec-
tiveness of the operating, control, and maintenance procedures.

B. Validation Life Cycle


1. Determination of Quality Attributes
In performing the validation, defining the quality attributes—that is, gaining a
clear understanding of the required quality and intended use—is the most impor-
tant issue, and should be determined before starting the validation. Without
defining required quality attributes we cannot establish validation protocols,
which are the basis of all validation studies.

2. The Validation Protocol


A validation protocol is defined as
A written plan stating how validation will be conducted and defining accep-
tance criteria. For example, the protocol for a manufacturing process identi-
fies process equipment, critical process parameters/operation ranges, prod-
uct characteristics, sampling, and test data to be collected, number of
validation runs, and acceptable test results [1].

The validation protocol is a detailed plan for conducting a validation


study. It is drafted by the individual or task group responsible for the project,
reviewed for content and completeness following the firm’s protocol review
procedure, and approved by designated individuals. It describes the responsibili-
ties of each individual or unit involved in the project.
All protocols, whether for IQ (installation qualification)/OQ (operational
qualification) of new equipment or for validating a new process, have the same

Copyright © 2003 Marcel Dekker, Inc.


basic format. They start with an objective section, which describes the reasons
for conducting the validation study as well as the results to be achieved. Next
there is a scope section. Here what is to be included and excluded from the
study is specified, effectively establishing the boundaries for the study.
Following the objective and scope sections is a detailed description of the
process/equipment to be validated. Here block diagrams of equipment, batch
formula and master manufacturing records, process flow diagrams, and other
documents that will help with the descriptive process are essential and should
be attached to the protocol. The protocol should contain a detailed description
of the sampling and testing schedule and procedures and clearly state the accep-
tance criteria for each stage of validation, such as DQ (design qualification), IQ,
OQ, and PQ (performance qualification). The number of times that specific
trials will be replaced in order to demonstrate reproducibility of results must be
specified.
The protocol should be endorsed by designated representatives of each unit
that will participate in the validation study. This is an essential step for validation
study. It should be described that the protocol is accepted by responsible persons,
and that each unit understands and agrees to fulfill its responsibilities as stated in
the protocol. Subsequent changes to the protocol, should they be necessary, must
be endorsed by the same individuals. Protocol addenda are sometimes necessary
because circumstances later arise that were impossible to anticipate when the study
was planned and the protocol drafted. In addition to approvals, the validation
protocol should have the appended data sheets, which are to be filled with data
obtained from the validation studies and compared with the criteria.

3. Steps of Validation
Validation plans for water and air systems typically include the following steps
(Fig. 1):
1. Establishing standards for quality attributes of water and air to man-
ufacture pharmaceuticals.
2. Defining systems and subsystems suitable to produce the desired
water and air by considering the quality grades of water and air.
3. Designing equipment, controls, and monitoring technologies.
4. Establishing standards for operating parameters of the selected
equipment of the system.
5. Developing an IQ stage consisting of instrument calibrations, inspec-
tions to verify that the drawings accurately depict the as-built config-
uration of the system, and special tests to verify that the installation
meets the design requirements. These items include pipe and instru-
ment drawings, air pressure differentials, air velocities, and airflow
patterns.

Copyright © 2003 Marcel Dekker, Inc.


Figure 1 Validation life cycle of water and air system. (From Ref. 2.)

Copyright © 2003 Marcel Dekker, Inc.


6. Developing an OQ stage consisting of tests and inspections to verify
that the equipment, system alerts, and controls are operating.
7. Establishing alert and action levels for the operational standards and
routine control. This phase of qualification may overlap with aspects
of the next step.
8. Developing a prospective PQ stage to confirm the appropriateness of
critical process parameter operating ranges. System reproducibility is
to be demonstrated in this stage over an appropriate time period.
During this phase of validation, alert and action levels for key qual-
ity attributes of water, such as TOC, pH, particulates and microbes,
and operating parameters for an air system (e.g., temperature, time,
air pressure differential, airflow velocity, and air exchange rate) are
verified.
9. Supplementing a validation maintenance program (also called con-
tinuous validation life cycle) that includes a mechanism to control
changes to the system and establishes and carries out scheduled pre-
ventive maintenance, including recalibration of instruments.
10. Instituting a schedule for periodic review of the system performance
and requalification.
11. Completing protocols and documenting steps 1 through 10 [2].

4. Control During Routine Operation


Revalidation and Change Control. Once the validation is completed, the
standard operating procedures (SOPs) are formalized. Routine operation should
be performed according to the established SOP.
Any proposed changes should be evaluated for their impact on the whole
system. The necessity for requalifying the system because of changes should be
determined. Revalidation and evaluation should be performed depending upon
the impact that might be caused by the change.
Alert and Action Levels. Validated and established systems should be
periodically monitored to confirm that they continue to operate within their
design specifications and consistently produce water or air of acceptable quality.
Monitored data may be compared to established process parameters or product
specifications. A refinement to the use of process parameters and product speci-
fications is the establishment of alert and action levels, which signal a shift in
process performance. Alert and action levels are distinct from process parame-
ters and product specifications in that they are used for monitoring and control
rather than accept or reject decisions. The levels should be determined based on
the statistical analysis of the data obtained by monitoring at the PQ step.
Alert levels are levels or ranges that when exceeded indicate that a process
may have drifted from its normal operation condition. Alert levels indicate a

Copyright © 2003 Marcel Dekker, Inc.


warning and do not necessarily require a corrective action. Exceeding an action
level indicates that corrective action should be taken to bring the process back
into its normal operating range [2,8].

III. QUALIFICATION OF WATER SYSTEMS


A. Required Quality for Water for
Pharmaceutical Purposes
Water is one of the most widely used substances, and raw materials, or an
ingredient in the production, processing, and formulation of pharmaceuticals.
Control of the organic and inorganic impurities and microbiological qual-
ity of water is important because proliferation of micro-organisms ubiquitous in
water may occur during the purification, storage, and distribution of this sub-
stance. Although there are various quality grades of water used for pharmaceuti-
cal purposes, all kinds of water are usually manufactured from drinking water
or comparable grade water as a source water.
Grades of water are closely related to the manufacturing methods and
distribution system of water. Major differences among these grades of water
consist of the following quality attributes:
Microbial counts
Endotoxin, which is due to the presence of microbes
Organic and inorganic impurities
Grades of water specified in the compendia (USP) are classified according
to the above quality attributes as
1. Potable water
2. Purified water
3. Water for injection
4. Sterile water for injection
5. Sterile water for inhalation
6. Sterile water for irrigation
7. Sterile bacteriostatic water for injection
Grades of water specified in the Pharmacopeia (USP) are summarized in
Table 1. “Water for injection” (WFI) is the most purified water, and careful
attention should be paid to the validation of its manufacturing process.

B. Selection of Water for Pharmaceutical Purposes


The quality attributes of water for a particular application are dictated by the
requirement of its usage. Sequential steps that are used for treating water for
different pharmaceutical purposes are shown in Figure 2 [6].

Copyright © 2003 Marcel Dekker, Inc.


Table 1 Specifications of Water for Pharmaceutical Use

Microbial Particulate
Inorganics control Microbial limit matter Endotoxin

Source water (I) −a −a −a −a −a


City water (potable) (II) Reg.b Reg.b Reg.b −a −a
Purified water (deionized) (III) +c +c 100 CFU/mLd −a −a
Purified water (membrane) (IV) +c +c 100 CFU/mLd −a +
WFI (rinse) (V) +c +c 100 CFU/mLd −a +
WFI (preparation) (VI) +c +c 100 CFU/mLd +f <0.25 EU/mle
WFI (LVP) (VII) +c +c 100 CFU/mLd +f <0.25 EU/mle
a
No control, no specifications.
b
City water regulations.
c
Controlled to be less than city water specifications. Microbial counts in deionized water should be carefully controlled.
d
According to the specifications by USP/EP, and recommended criteria final rinse water: 10 CFU/100 mL.
e
Cooling water used for sterile products: 1 CFU/100 mL.
f
Particulate matter for LVP: (>10 µm, 20/ml), >25 µm, 2/ml).

Copyright © 2003 Marcel Dekker, Inc.


Figure 2 Water for pharmaceutical purposes. (From Ref. 2.)

The manufacturing method and distribution system also have a close rela-
tion with the construction design of facilities and equipment.
1. Selection of the most suitable quality grade of water for its intended use.
2. Determination of the water manufacturing system elements, including
facility and equipment.
3. Design of water manufacturing system, including the design of system
equipment.
4. After construction of the water system is completed based on its de-
sign, the system has to be scrutinized as to whether it has been built
to design specification or not.

Copyright © 2003 Marcel Dekker, Inc.


5. After confirming the installation of facility and equipment, the quality
of water produced is examined from various viewpoints according to
the predetermined specifications.
6. In the routine production of water, representative quality items of wa-
ter have to be monitored to confirm the performance of normal opera-
tion, and if any undesirable trends or out of specification values are
found, corrective action should be taken.
The steps of checking design and construction, confirming proper installa-
tion and operation, and documenting these processes are collectively called qual-
ification or validation. In case of any system change or changes to equipment,
the same kinds of procedures should be implemented.
Water for pharmaceutical purposes, and selection of water grade for phar-
maceuticals are summarized in Figures 1 and 2. Specifications of various kinds
of water are summarized in Table 1.
Major items of quality attributes that should be controlled and specified
for pharmaceutical use are
1. Organic impurities
2. Inorganic impurities
3. Particulates
4. Microbes
5. Endotoxin

C. Design Qualification of Water Systems


The quality attributes of water for a particular application are dictated by the
requirements of its usage. Production of pharmaceutical water employs a combi-
nation of sequential unit operations (processing steps) that address specific water
quality attributes.
The validation plan should be designed to establish the suitability of the
system and provide a thorough understanding of the purification mechanism,
range of operating conditions, required pretreatment, and the most likely mode
of failure. It is also necessary to demonstrate the effectiveness of the monitoring
scheme and to establish the requirements for validation maintenance. The selec-
tion of specific unit operations and design characteristics for a water system
should take into consideration the quality of the feed water, the technology
chosen for subsequent processing steps, the extent and complexity of the water
distribution system, and the appropriate requirements. In a system for WFI, the
final process (distillation, reverse osmosis, or ultrafiltration) must have effective
bacterial endotoxin reduction capability and must be validated for each specific
equipment unit. The final unit operations used to produce WFI have been lim-
ited to distillation, reverse osmosis, and/or ultrafiltration. Distillation has a long

Copyright © 2003 Marcel Dekker, Inc.


history of reliable performance for the production of WFI. Other technologies,
such as reverse osmosis and ultrafiltration, may be suitable in the production of
WFI if they are appropriately validated for each specific set of equipment.
Typical sequential processing steps that are used for manufacturing puri-
fied water (PW) are shown in Figure 3. A distilled water distribution system
that is commonly used for WFI is shown in Figure 4.
1. Step 1 is the combination of prefilter, carbon filter, and ion exchanger
(softener). After chlorine is removed, attention has to be paid to pre-

Figure 3 Typical sequential processing steps for water treatment.

Copyright © 2003 Marcel Dekker, Inc.


Figure 4 Typical sequential processing steps for distilled water treatment. When cool-
ing water is not needed, water will circulate at 80°C or above. During use the cooling
water valve is open and the loop outlet valve is closed. After use the loop outlet valve
is kept closed and the loop drain valve opened to flush all the cooled water to drain.

vent microbial growth. For this purpose, ultraviolet light is installed


and the water is circulated.
2. Step 2 is a reverse osmosis process, after adjusting temperatures by
heat exchanger.
After the reverse osmosis process, inorganic impurities are completely
removed by anion exchanger and cation exchanger. An ultraviolet light is in-
stalled and the water is circulated to prevent microbial growth.
The obtained water can be used for nonparenteral dosage forms. For the
parenteral purpose, water obtained in this way is usually distilled.
Water for injection obtained by distillation is circulated through the main
loop and subloop at 80. During use the cooling water valve is open and the loop
outlet valve is closed. After use the loop outlet valve is kept closed and the loop
drain valve is opened to flush all the cooled water to drain.
A typical evaluation process to select an appropriate water quality for a
particular pharmaceutical purpose is shown in the decision tree in Figure 5 [2].

D. Qualification of Equipment and Components


for Water System
Equipment and components used for the water system must maintain sanitary
integrity and be anticorrosive and assured for technical integrity.

Copyright © 2003 Marcel Dekker, Inc.


Figure 5 Selection of water for pharmaceutical purpose. *, Water for sterile BPCs or
dosage forms must be rendered sterile if there is not a sterilization step following addi-
tion; ‡, microorganism control can occur either in water treatment or in BPC process; †,
endotoxin removal can occur either in water treatment or in BPC process; §, NPDWR
water—water meeting EPA national primary drinking water regulations. (From Ref. 2.)

Copyright © 2003 Marcel Dekker, Inc.


1. Components
Selection should be made with assurance that it does not create a source for
contamination intrusion.

2. Piping and Installation


Stainless steel welds should provide reliable joints that are internally smooth
and corrosion-free. Low carbon stainless steel (SUS 304L, 316, and 316L) com-
patible wire filler, and where necessary, inert gas, automatic welding machines,
and regular inspection and documentation, help to ensure acceptable weld qual-
ity. Follow-up cleaning and passivation are important for removing contamina-
tion and corrosion products and to re-establish the passive corrosion-resistant
surface. Piping systems should be installed and sloped in such a way that they
are completely self-draining. Complete drainage is important, as it prevents the
formation of standing “pools” of liquid that can support the growth of microbes.
Further, properly sloped piping prevents the formation of condensate “plugs”
that can cause cold spots during SIP, and most important it allows for the free
drainage of all rinsing and washing solutions during CIP, which enhances clean-
ing efficiency. The slope is normally 1/200 to 1/100. Isometric drawings of
piping systems for water systems are essential for the design and installation
qualification of both water supply and CIP/SIP piping systems.
3. Material
Materials of construction should be selected to be compatible with mater-
ials used as control measures, such as sanitizing, cleaning, and passivating
media.
Plastic materials can be fused (welded) in some cases and also require
smooth, uniform internal surfaces. Adhesives should be avoided due to the po-
tential for voids and chemical reactions. Mechanical methods of joining, such
as flange fittings, require care to avoid the creation of offsets, gaps, penetrations,
and voids. Control measures include good alignment, properly sized gaskets,
appropriate spacing, uniform sealing force, and the avoidance of threaded fit-
tings.
Temperature rating is a critical factor in choosing appropriate materials
because surfaces may be required to handle elevated operating and sanitiza-
tion temperatures. Should chemicals or additives be used to clean, control, or
sanitize the system, materials resistant to these chemicals or additives must be
utilized.
4. Surface Polishing
The finish on metallic materials such as stainless steel, whether it be a refined
mill finish, polished to a specific grit, or an electropolished treatment, should

Copyright © 2003 Marcel Dekker, Inc.


complement system design and provide satisfactory corrosion and microbial ac-
tivity resistance.

5. Dead Legs
Dead legs pose two problems for CIP. First, cleaning fluids must be able to
flush out trapped gas pockets in order to wet all the piping surfaces in the dead
legs. Second, fresh cleaning fluid must flush the dead leg to maintain rapid
cleaning rates. Dead legs should not be greater in length than six diameters (6D)
of the unused portion measured from the axis of the pipe in use.

6. Valves
The most commonly used valves in process piping systems for PW and WFI
used for pharmaceutical manufacturing are diaphragm valves. This is because
they are easily CIP-cleanable and provide complete containment of in-process
materials. Diaphragm valves are limited in the ways they may be installed for
free drainage; they sometimes are prone to leakage and they have a relatively
high pressure drop as compared with other types of valves.
For situations in which complete containment is not required, “plunger-
type” compression valves of hygienic design may be used. These have several
advantages over diaphragm valves regarding installation and operation but they
do not provide complete containment.
Ball and butterfly valves are also commonly used in water treatment sys-
tems. Diaphragm valves should be used downstream from the unit that removes
dissolved solids (reverse osmosis unit or deionizer), however, because of their
inherent ease of sanitation.

7. Pumps
Pumps should be of sanitary design with seals that prevent contamination of the
water. Pumps moving water for manufacturing or final rinsing, water for cooling
the drug product after sterilization, and in-process or drug product solutions
shall be designed to utilize water for injection as a lubricant for the seals. Sev-
eral types of CIP-cleanable pumps are commonly used in water systems or phar-
maceutical manufacturing processes. These include centrifugal, rotary lobe, peri-
staltic, and diaphragm pumps, of which all but the centrifugal pump provide
positive displacement.

8. In-Line Instrumentation
In-line instruments or sensors are necessary components for automated pro-
cesses. For ease of cleaning, sensors should be chosen that directly mount onto
vessel nozzles or piping tees with minimum dead leg distances. Also, the instru-

Copyright © 2003 Marcel Dekker, Inc.


ments should be of a cleanable design and constructed to similar standards as
those for process equipment and piping.

9. Pressure Gauges
Sanitary diaphragm-style pressure gauges should be used when possible, as they
are very cleanable. When pressure gauges are installed in process piping, the
diameter should be less than 6D.

10. Heat Exchangers


Heat exchangers should be double tube sheet or concentric tube design. Heat
exchangers other than the welded double-concentric tube type or double-tube
sheet type must employ a pressure differential and a means for monitoring the
differential. The pressure differential shall be such that the fluid requiring a
higher microbial quality shall be that with the greater pressure.

11. Distillation
Distillation equipment is used to produce USP WFI-quality water. The distilla-
tion process removes dissolved solids not otherwise removed by deionizers or
RO units.
The chemical quality of the steam supplied to the still must be controlled
to prevent recontamination of the distillate. Also, the condenser must be of a
double-tube design to prevent condenser coolant from coming into direct contact
with the distallate, thereby causing recontamination.
Separation of mists by the distillation process is important to remove the
endotoxin or other contaminant. Distillators have their own upper limits of
throughput capacity. Usually the more the amount of water generated at the unit
period, the more the conductivity increases at the range of maximum capacity.
This means that the purity of the water becomes worse.

12. Filters
Final filters of water for injectable products may not be used at any point in the
piping system of water for manufacturing or final rinse.
Water storage tank vent filters must be equipped with a sterilizing air filter
in order to prevent the air, which displaces water drawn from the tank, from
microbiologically contaminating the water. The filter must be hydrophobic in
order to prevent condensation from blinding the filter and preventing air entry
or escape from the tank. It also must have a mean porosity of less than 1 µm.
Water filters are used in various locations in water treatment systems for
two basic purposes: removal of undissolved solids, some of which are added to
the water by various components of the water treatment system, and removal of

Copyright © 2003 Marcel Dekker, Inc.


bacterial contaminants. Filters are commonly used downstream from carbon
beds and resin beds and on the incoming water supply line, and they are typi-
cally in the order of 10 to 15-µm mean porosity. Membrane filters of 0.2 µm
are used to remove bacteria. Filters must be properly maintained in order to
keep the water treatment system operating efficiently and to prevent them from
becoming a source of bacterial and endotoxin contamination.
Microbes are not destroyed by bacteria-retentive filters but instead become
concentrated in and on them. Certain bacteria have the capability of growing
through a membrane filter. Also, filters can become damaged by frequent or
sudden changes in water pressure (water hammer).
13. Deionizers
These devices are used to remove dissolved solids from the feed water. Dioniz-
ers use ion exchange resins to remove charged particles. Cation resin beds re-
move negatively charged particles; anion resins remove positively charged parti-
cles. Mixed bed deionizers (containing both cation and anion exchange resins)
are commonly used to give the water a final “polishing” treatment. Resins lose
their ability to remove charged particles and must be periodically regenerated
using strong caustic and acid solutions. This treatment also sanitizes the resin
beds, which, like carbon beds, are a fertile breeding ground for bacteria when
improperly maintained.
14. Auxiliary Equipment
1. Backflow of liquids shall be prevented at points of interconnection of
different systems.
2. Pipelines for the transmission of water for manufacturing or final
rinse and other liquid components shall be constructed of welded
stainless steel (nonrusting grade) equipped for sterilization with
steam, except that sanitary stainless steel lines with fittings capable
of disassembly may be immediately adjacent to the equipment or
valves that must be removed from the lines for servicing and replace-
ment.
3. Auxiliary equipment and fittings that require seals, gaskets, dia-
phragms, filter media, and membranes should exclude materials that
permit the possibility of extractables entry, shedding, and microbial
activity [7–12].
15. Ultraviolet Light
Ultraviolet light (UV) is not effective enough to eliminate existing biofilm.
When coupled with conventional thermal or chemical sanitization technologies,
however, it is most effective and can prolong the interval between system sani-
tizations.

Copyright © 2003 Marcel Dekker, Inc.


The use of UV light also facilitates the degradation of hydrogen peroxide
and ozone. The most effective biocidal wavelength is 253.7 nm. The amount of
light at 255 nm emitted by a UV light decreases with time, so lamps have to be
monitored and replaced when necessary.

16. Wastewater
Waste liquids shall be introduced to sewers through trapped drains. Drains from
equipment shall be designed with an atmospheric break to prevent back-
siphonage.
All stills and tanks holding liquid requiring microbial control shall have
air vents with non-fiber-releasing sterilizable filters capable of preventing micro-
bial contamination of the contents. Such filters shall be designed and installed
so that they do not become wet. Filters shall be sterilized and installed aseptic-
ally. Tanks for holding water require air vents with filters [7,10].

E. Sanitization
Microbial control in water systems is achieved primarily through sanitization
practices. Systems can be sanitized using either thermal or chemical means. In-
line UV light can also be used to “sanitize” water in the system continuously.

1. Thermal Approaches
Thermal approaches to system sanitization include periodic or continuously cir-
culating hot water and the use of steam. These techniques are limited to systems
that are compatible with the higher temperatures needed to achieve sanitization,
such as stainless steel and some polymer formulations. Hot water circulation is
effective or essential for this purpose, especially for the WFI system.

2. Chemical Methods
Chemical methods, where compatible, can be used on a wider variety of con-
struction materials. These methods typically employ oxidizing agents such as
hypochlorite, hydrogen peroxide, ozone, or per-acetic acid. Hypochlorites are
effective sanitizers but are difficult to flush from the system and tend to leave
biofilms intact.

3. Validation of Sanitization Steps


Sanitization steps require validation to demonstrate the capability of reducing
and holding microbial contamination at acceptable levels. Validation of thermal
methods should include a heat distribution study to demonstrate that sanitization
temperatures are achieved throughout the system. Validation of chemical meth-

Copyright © 2003 Marcel Dekker, Inc.


ods requires a demonstration of adequate chemical concentrations throughout
the system. In addition, when the sanitization process is completed, effective
removal of chemical residues must be demonstrated.
The frequency of sanitization is generally dictated by the results of system
monitoring. Conclusions derived from the trend analysis of the microbiological
data should be used as the alert mechanism for maintenance. The frequency
of sanitization should be established so that the system operates in a state of
microbiological control and does not exceed alert levels.

4. SIP/CIP
For a WFI or highly purified water system, the SIP/CIP method is usually ap-
plied. In an SIP/CIP system, sterilization and/or cleaning take in place in situ or
without dissembling the equipment. Consequently, the result of sterilization and/
or cleaning is confirmed by the process parameters previously validated. Based
on process parameters and their ranges previously determined by the process
validation, the SIP/CIP process can be confirmed whether or not it is completely
sterilized and cleaned. This is a typical application of the concept of validation.

5. Hot Water Circulation of WFI System


Hot water circulation systems circulate hot water through all pipelines from the
storage tank through all use points to return to the storage tank. By a combina-
tion of hot water circulation system and SIP, microbial quality of WFI can be
guaranteed to be 0 cfu per 100 ml. Once the water in the system is drained out,
the entire system must be steam-sterilized. By applying hot water circulation
and SIP, formation of any biofilm can be prevented if the piping and installation
are well designed and there are no dead legs [2,7–9,11,12].

F. Sampling Considerations
Water systems should be monitored at a frequency that is sufficient to ensure
that the system is in control and continues to produce water of acceptable qual-
ity. Samples should be taken from representative locations within the processing
and distribution system. Established sampling frequencies should be based on
system validation data and should cover critical areas. Unit operation sites might
be sampled less frequently than point-of-use sites. The sampling plan should
take into consideration the desired attributes of the water being sampled. Be-
cause of their more critical microbiological requirements, systems for WFI may
require a more rigorous sampling frequency.
When sampling water systems, special care should be taken to ensure that
the sample is representative. Sampling ports should be sanitized and thoroughly
flushed before a sample is taken. Samples for microbiological analysis should

Copyright © 2003 Marcel Dekker, Inc.


be tested immediately or suitably protected to preserve the sample until analysis
can begin. The sampling operation itself might often cause a microbial contami-
nation.

1. Biofilm, Planktonic Micro-Organisms, and Benthic


Micro-Organisms
Samples of flowing water are only indicative of the concentration for planktonic
(free-floating) micro-organisms present in the system. The number of microbes
determined by the water system monitoring is an indication of floating microbes
in water; that is, planktonic micro-organisms. Benthic (attached) micro-organ-
isms in the form of biofilms are generally present in greater numbers and are
the source of the planktonic population.
The major purpose of monitoring microbes is to identify the generation of
biofilms and to find the locations of biofilms, if any. The purpose of sanitization
is to kill and destroy the biofilm after detecting the location of the biofilms. The
planktonic population, whose number of micro-organisms in water is monitored,
should be understood and utilized to indicate biofilms in the system. The num-
ber of microbes in water is an indicator of system contamination levels and is
the basis for the system alert levels.
If there were no microbials in water, there would not be any biofilms in
the system. If any microbials are detected in the system, there must be biofilms
in some locations. Biofilm is formed in stagnant places, such as valves, dead
ends, or unsuitably sloped piping. Detecting micro-organisms and biofilms is
one method of controlling the cleanliness of the system. The other method is to
completely eliminate the stagnant places or dead ends that might cause biofilms.
From such viewpoints, the design and construction of a desirable water system
as described in sec. III.D is the fundamental way to prevent the formation of
biofilms, and consequently to both reduce the number of micro-organisms and
prevent the generation of micro-organisms in the system.

2. Operation, Monitoring, and Control


Monitoring programs should be established to ensure that the water system re-
mains in a state of control. The program should include
1. Procedures for operating the system
2. Monitoring programs for critical quality attributes and operating con-
ditions, including calibration of critical instruments
3. Schedule for periodic sanitization
4. Preventive maintenance of components
5. Control of changes to the mechanical system and to operating condi-
tions

Copyright © 2003 Marcel Dekker, Inc.


3. Operating Procedures
Procedures for operating the water system and performing a routine monitoring
program should be established based on the validation study. The procedures
should be well documented, detail the function of each job, assign who is re-
sponsible for performing the work, and describe how the job is to be conducted.

4. Monitoring Program
Critical quality attributes and operating parameters should be documented and
monitored. The program may include a combination of in-line sensors or record-
ers (e.g., a conductivity meter and recorder), manual documentation of opera-
tional parameters (such as carbon filter pressure drop), and laboratory tests (e.g.,
total microbial counts). The frequency of sampling, the requirement for evaluat-
ing test results, and the necessity for initiating corrective action should be in-
cluded.

G. Microbial Considerations
The major exogenous source of microbial contamination is source or feed water.
At a minimum, feed water quality must meet the quality attributes of potable
water for which the level of coliforms is regulated. A wide variety of other
micro-organisms, chiefly gram-negative bacteria, may be present. These micro-
organisms may compromise subsequent purification steps. Examples of other
potential exogenous sources of microbial contamination include unprotected
vents, faulty air filters, backflow from contaminated outlets, drain air breaks,
and replacement activated carbon and deionizer resins. Sufficient care should
be given to system design and maintenance in order to minimize microbial con-
tamination from these sources.
Micro-organisms present in feed water may adsorb to carbon beds, deion-
izer resins, filter membranes, and other unit operation surfaces and initiate the
formation of a biofilm [2,8].

H. Endotoxin
Endotoxins are lipopolysaccharides from the cell envelope that is external to the
cell wall of gram-negative bacteria. Gram-negative bacteria readily form biofilm
that can become a source of endotoxin. Endotoxin may be associated with living
micro-organisms or fragments of dead micro-organisms, or may be free molecules.
The free form of endotoxin may be released from cell surfaces or biofilm that
colonize the water system, or they may enter the water system via the feed water.
Endotoxin should be eliminated by means of distillation, reverse osmosis,
and/or ultrafiltration. Generation of endotoxin is prevented by controlling the

Copyright © 2003 Marcel Dekker, Inc.


introduction of micro-organisms and microbial proliferation in the system. This
may be accomplished through sanitization and sterilization.
The presence of endotoxin should be monitored by LAL method in the
routine operation. Endotoxin can be removed by means of distillation, reverse
osmosis, and/or ultrafiltration. Incomplete separation of mist in distillation, how-
ever, and leakage in membrane of reverse osmosis or ultrafiltration cause con-
tamination with endotoxin. After these separation processes, of course, contami-
nation of microbes or growth of microbes causes endotoxin contamination [2,8].

I. Methodological Considerations
The objective of a water system microbiological monitoring program is to pro-
vide sufficient information to control the microbiological quality of the water
produced. An appropriate level of control may be maintained by using data
trending techniques and limiting specific contraindicated micro-organisms, con-
sequently it may not be necessary to detect all of the micro-organisms present.
The methods selected should be capable of isolating the numbers and types of
organisms that have been deemed significant relative to system control and
product impact for each individual system [2,8].

J. Continuous Automatic Monitoring of Water


Monitoring and feeding back the data are important in maintaining the perfor-
mance of water systems. By applying an automatic continuous monitoring sys-
tem combined with the method of trend analysis, processes can be maintained
in a much more stable state. For example, this can be achieved by applying
automatic continuous monitoring of TOC and conductivity of the water system.
TOC and conductivity are the major quality attributes of water by which
organic and inorganic impurities can be determined.

IV. QUALIFICATION OF AIR HANDLING SYSTEMS


A. Purposes of an Air Handling System
The purposes of an air handling system are
1. To prevent microbial contamination of sterile products and of clean
areas
2. To prevent the spreading and contamination of virus, pathogenic, and
spore-forming microbes used in the manufacturing of pharmaceuticals
3. To prevent spread and contamination of penicillin or other sensitizing
drugs, cytotoxic drugs, and drugs with strong pharmacological action

Copyright © 2003 Marcel Dekker, Inc.


4. To prevent cross-contamination of solid dosage form or bulk pharma-
ceuticals, whose fine powder tends to spread and disperse
CFR211.46 states that “a) Adequate ventilation shall be provided.
b) Equipment for adequate control over air pressure, micro-organisms, dust,
humidity, and temperature shall be provided when appropriate for the manufac-
ture, processing, packaging, or holding of a drug product. c) Air filtration sys-
tem, including prefilters and particulate matter air filters, shall be used when
appropriate on air suppliers to production areas.”
In the air handling system, special attention has to be paid to keep the
environment clean and to prevent the contamination of products. There are two
different kinds of concepts to control the air system: one is to prevent intrusion
of the surrounding air (positive air pressure control), and the other is for the
containment of air containing an undesirable substance generated in the opera-
tion area (negative air pressure control). Air handling systems should be de-
signed, installed, and maintained to meet these purposes.

B. The Concept of Air Handling System Validation


The degree of cleanliness of air in the pharmaceutical manufacturing and related
operation area should be established depending on the characteristics of products
and operations in the area. In order to establish and maintain such standards,
careful attention has to be exercised to keep the standards from the stage of design
and construction through to the monitoring in the stage of routine operations.
A total air handling system, covering the open air intake, treatment, the
supply to the manufacturing area, and the exhaust, should be designed and vali-
dated. The handling system contains units of prefiltration, temperature and hu-
midity control, final air filtration, return, and exhaust. When the air is supplied
to the manufacturing area, care is required in maintaining the required air quality
during the operation or at the point of product exposure to the environment.
This point is closely related to the layout and construction features of the manu-
facturing area.
1. The air must flow from the critical or most clean area to the surround-
ing area; that is, the less clean area. For this purpose, rooms used for
the manufacturing operation have to be laid out according to the order
of the required air cleanliness.
2. In order to maintain the air cleanliness in the area and airflow, the
amount of air supplied and exhaust have to be balanced to keep the
designed air exchange ratio, airflow pattern, and air pressure differen-
tials. In each room the operation site should be maintained in the
most suitable status. For such purposes, the following items must be
carefully controlled:

Copyright © 2003 Marcel Dekker, Inc.


a. Locations and number of air supplies
b. Locations and number of air exhausts
c. Ratio of air exchange
d. Return ratio of exhaust air
e. Location of local air exhaust, if necessary
f. Airflow pattern at the site of product exposure
g. Air velocity at the point of product exposure
These features have to be well designed, installed, validated, and main-
tained. Critical operation has to be performed under the unidirectional airflow
(laminar airflow). Air turbulence deteriorates air quality by intake of air from
the surrounding less clean areas.
The amount of air supplied and exhausted is related to the air pressure
differentials. After the system is validated, air quality should be continuously
monitored and maintained during manufacturing operations.
Filters used for the prefiltration and final filtration should be maintained
to operate to their design specifications. Deterioration of filters is caused by
leakage and/or accumulation of particles. The former is tested by periodical
integrity test (usually dioctylphthalate DOP test), and the latter is tested by the
increase of air pressure differentials between the upstream and downstream sides
of the filter.

C. Validation of Air Handling Systems


All of the environmentally-controlled areas of pharmaceutical manufacturing
and its related areas should meet the requirement of air cleanliness, which is
expressed as classifications specified by official standards, such as ISO (Interna-
tional Organization of Standardization) or FED-STD (U.S. federal standard)
209, and/or GMP. The classification has a close relationship with the air treat-
ment procedures and construction features.
1. First of all, the most suitable quality grade of air for the manufactur-
ing environment, and operation performed has to be selected.
2. Second, the air handling system/method that suits the facility and
combination of equipment has to be designed. Therefore, design qual-
ification is the first step of the validation.
3. Before completing construction of the air handling system, the con-
structed system has to be scrutinized as to whether or not it is built
according to the design.
4. After confirming the installation of facility and equipment, the quality
of air is examined from various viewpoints according to the predeter-
mined specifications.
5. In the routine operation, representative quality items have to be moni-

Copyright © 2003 Marcel Dekker, Inc.


tored to confirm the performance of normal operation, and if any
undesirable trends or out of specification results are found, corrective
action should be taken. These processes of checking design and con-
struction, confirmation of proper installation and operation, and docu-
mentation of these processes are termed qualification/validation.
6. In case of system change or any changes of equipment, the same
procedure should be taken. These processes are summarized as fol-
lows:
a. Selection of air quality
b. Determination of air handling system and design of the construc-
tion features
c. Construction and qualification of installation
d. Test run and operational qualification
e. Operation of air handling system and confirmation of the air qual-
ity; that is, qualification of operation, and monitoring of opera-
tions; that is, operational qualification
f. Revalidation, or change control [9,10,12]

D. Classification of Air Quality and Design Qualification


1. Establishment of Clean Room Classifications
The design and construction of clean rooms and controlled environments are
specified in USP, FED-STD 209E, and ISO air cleanliness standards. The clean-
liness classifications are defined by the absolute concentration of airborne parti-
cles. Methods used for the assignment of air classification of controlled environ-
ments and for monitoring airborne particulates are included in these standards.
FED-STD 209E or ISO standards of air cleanliness and controlled environments
are used by pharmaceutical manufacturers to provide specifications for clean
rooms and the commissioning and maintenance of these facilities. Data available
in the pharmaceutical industry, however, provide no scientific agreement on a
relationship between the number of nonviable particulates and the concentration
of viable micro-organisms. Nevertheless, the rationale that the fewer particulates
present in a clean room the less likely it is that airborne micro-organisms will
be present is accepted and can provide pharmaceutical manufacturers and build-
ers of clean rooms and other controlled environments with engineering standards
in establishing a properly functioning facility.
As applied in the pharmaceutical industry, FED-STD 209E and ISO Air
Cleanliness Standards are based on limits of all particles with sizes equal to or
larger than 0.5 µm. Table 2 describes airborne particulate cleanliness classes
in federal standard 209E and ISO air cleanliness standards as adapted to the
pharmaceutical industry. It is generally accepted that if fewer particulates are

Copyright © 2003 Marcel Dekker, Inc.


Table 2 Air Quality Classification and Concentrations of Controlled Environment

U.S. EEC/CGMP
Particles per Particles per m3 ISO classification of 209E USP 209E (1989)/WHO
m3 >0.1 µm ⭌ 0.5 µm airborne particulates (1992) customary GMP

— 1.00 —
102 3.50 ISO class 2 — — —
— 1.00 — M1 — —
103 35.30 ISO class 3 M1.5 1 —
— 102 — M2 —
104 3.53 × 102 ISO class 4 M2.5 10 —
— 103 — M3 — —
105 3.53 × 103 ISO class 5 M3.5 102 A&B
— 104 — M4 — —
106 3.53 × 104 ISO class 6 M4.5 103 —
— 105 — M5 — —
107 3.53 × 105 ISO class 7 M5.5 104 C
— 106 — M6 — —
108 3.53 × 106 ISO class 8 M6.5 105 D
— 107 — M7 —

Source: Refs. 15, 19.

present in an operational clean room or other controlled environment, the less


the microbial count under operational conditions.
Clean rooms are maintained under a state of operational control on the
basis of dynamic (operational) data. Class limits are given for each class name.
The limits designate specific concentrations (particles per unit volume) of air-
borne particles with sizes equal to and larger than the particle sizes shown in
Table 2 [7,10–12,14].
Air quality relating to the manufacturing of sterile pharmaceutical prod-
ucts is designated in WHO and EU GMP as A, B, C, and D, and in USP as
class 100, class 10,000, and class 100,000. These classes correspond to ISO
class 5, ISO class 7, and ISO class 8, respectively (there is no class correspond-
ing to B grade in FDA/USP) [13–19].

2. ISO Classification of Air Cleanliness


The ISO air cleanliness level (class) is expressed in terms of an ISO air classifi-
cation number (class N). This represents the maximum allowable concentrations
(in particles/quantity of air) for considered sizes of particles [18,19]. The con-
centrations are determined by using the formula given below.

Copyright © 2003 Marcel Dekker, Inc.


Airborne particulate cleanliness shall be designated by a classification
number N. The maximum permitted concentration of particles Cn for each con-
sidered particle size D is determined from the formula

冉 冊
2.08
0.1
Cn = 10N ×
D

where

Cn represents the maximum permitted concentration (in particles/m3 of


air) of airborne particles that are equal to or larger than the considered
particle size. Cn is rounded to the nearest whole number, using no
more than three significant figures.
N is the ISO classification number, which shall not exceed a value of 9.
D is the considered particle size in µm.
0.1 is a constant with a dimension of µm.

Figure 6 presents relationships between sizes of airborne particulates and


concentrations in each ISO air cleanliness class. The relationship between the
requirement for air cleanliness and manufacturing operation is summarized in
Table 3.
Aseptic processing and processes related to sterile products manufacturing
should be carried out in the environment of the area under the defined air
quality.
Airflow should also be designed, validated, and confirmed to be main-
tained as such by the monitoring of air quality. There are no official require-
ments for the manufacturing of nonsterile products; however, air quality and
airflow should be designed, validated, and monitored for the purpose of prevent-
ing contamination.

E. Unidirectional Airflow (Laminar Flow)


Control Equipment
Area A (class 100, ISO class 5), which applies to air handling equipment at the
filling line and microbiological testing area, shall provide HEPA-filtered lami-
nar-flow air. (Note: The term laminar flow has not been used recently; instead
the term “unidirectional air flow” is used [FED-STD-209E, Sept. 11, 1992].
Unidirectional airflow [referred to as laminar airflow] is an airflow having gen-
erally parallel streamlines, operating in a single direction, and with uniform
velocity over its cross section [15,19].
Such equipment shall

Copyright © 2003 Marcel Dekker, Inc.


ISO c
lass
ISO c 9
lass
ISO c 8
lass
ISO c 7
lass
6
ISO c
lass
ISO c 5
lass
ISO c 4
lass
3
ISO c
lass
2
ISO c
lass
1

Figure 6 ISO classification of airborne particulate cleanliness. (From Ref. 19.)

1. Have hood or airflow direction panels and working surface areas that
are constructed of a smooth, durable, nonflaking material, such as
glass, plastic, or stainless steel.
2. Have prefilters that are disposable or fabricated from a material that
can be properly cleaned and reused.
3. Have HEPA final filters that have been tested to assure leak-proof
construction and installation.
4. Provide a laminar airflow with an average velocity of 90 ft per min
over the entire air exit area. The air velocity should be high enough
to maintain the unidirectional flow pattern.
5. Be monitored according to a written program and scheduled for com-
pliance with the requirements.
Schematic construction features for an aseptic processing area are shown
in Figure 7.

Copyright © 2003 Marcel Dekker, Inc.


Table 3 Air Quality Classification and Process Step

Products for European supply

Terminally sterilized

Typical process step Not unusually at risk Unusually at risk Aseptically processed

Dispensing Grade D Grade C (controlled) Grade C (controlled)a


Compounding Grade D Grade C (controlled) Grade C (controlled)a
Filtration From grade D to From grade C (con- From grade C (con-
grade C (controlled) trolled) to grade A trolled) to grade A
(critical) or closed (critical) [back-
systems ground grade B
(clean)] or closed
systems
Container prep/wash + Grade D Grade C (controlled) Grade D
stopper prep/wash
Container sterilization From grade D to From grade C (con- From grade D to
Depryogenation grade C (controlled) trolled) to grade A grade A (critical)
(critical)
Stopper Sterilization From grade D to From grade C (con- From grade D to
grade C (controlled) trolled) to grade A grade A (critical)
(critical)
Filling and stoppering Grade C (controlled) Grade A (critical) Grade A (critical)
[background grade [background grade
C (controlled)] B (clean)]
Lyophilization — — Grade A (critical)
[background grade
B (clean)]

Note. Capping and crimping, terminal sterilization, inspection and labeling and packaging “pharmaceuticals.”
a
For European aseptically produced products with sterile raw materials, where sterile filteration is not carried out,
then dispensing and compounding shall be in a grade A area, with a grade B background.
Source: Refs. 14, 20.

F. Performance Qualification and Parameters


of Cleanliness
A controlled environment such as a clean zone or clean room is defined by
certification according to a relevant clean room operational standard. Parameters
that are evaluated include
1. Number of airborne particles
2. Number of airborne microbes
3. Filter integrity, including HEPA filter leak test

Copyright © 2003 Marcel Dekker, Inc.


Figure 7 Major construction features for aseptic processing. (From Ref. 12.)

4. Air velocity
5. Airflow patterns
6. Air changes ratio
7. Pressure differentials
These parameters can affect the microbiological bioburden of the clean room.
Proper testing and optimization of the physical characteristics of the clean room
or controlled environment is essential prior to completion of the validation of
the microbiological monitoring program. Assurance that the controlled environ-
ment is operating adequately and according to its engineering specifications will
give a higher assurance that the bioburden of the environment will be appro-
priate for aseptic processing.

G. Microbiological Evaluation Program for


Controlled Environments
Airborne micro-organisms are not free-floating or single cells, but they fre-
quently associate with particles of 10 to 20 µm. Particulate counts as well as
microbial counts within controlled environments vary with the sampling loca-
tion and the activities being conducted during sampling.
Microbial monitoring programs for controlled environments should assess
the effectiveness of cleaning and sanitization practices by and of personnel that

Copyright © 2003 Marcel Dekker, Inc.


could have an impact on the bioburden of the controlled environment. Microbial
monitoring will not quantitate all microbial contaminants present in these con-
trolled environments. Routine microbial monitoring should provide sufficient
information to ascertain that the controlled environment is operating within an
adequate state of control, however.
Environmental microbial monitoring and analysis of data by qualified per-
sonnel will permit the status of control to be maintained in clean rooms and
other controlled environments. The environment should be sampled during nor-
mal operations to allow for the collection of meaningful data. Microbial sam-
pling should occur when materials are in the area, processing activities are ongo-
ing, and a full complement of operating personnel is on site.
When appropriate, microbial monitoring of clean rooms and some other
controlled environments should include quantitation of the microbial content of
room air, compressor air that entered the critical area, surfaces, equipment, saniti-
zation containers, floors, walls, and personnel garments (e.g., gowns and gloves).
The objective of the microbial monitoring program is to obtain representa-
tive estimates of the bioburden of the environment. When data are compiled and
analyzed, any trends should be evaluated by trained personnel. While it is im-
portant to review environmental results on the basis of recommended and speci-
fied frequency, it is also critical to review results over extended periods to deter-
mine whether or not trends are present. Trends can be visualized through the
construction of statistical control charts that include alert and action levels. The
microbial control of controlled environments can be assessed in part on the basis
of these trend data. Periodic reports or summaries should be issued to alert the
responsible manager [13].

H. Training of Personnel
The major source of microbial contamination of controlled environments is per-
sonnel. Since the major threat of contamination of product being aseptically
processed comes from the operating personnel, the control of microbial contami-
nation associated with these personnel is one of the most important elements of
the environmental control program. Personnel training should be conducted be-
fore the qualification and validation practice [13].

I. Sampling and Test of Air Quality


1. Critical Factors Involved in the Design and Implementation
of a Microbiological Environmental Control Program
An environmental control program should be capable of detecting an adverse
drift in microbiological conditions in a timely manner that would allow for
meaningful and effective corrective actions. An appropriate environmental con-
trol program should include identification and evaluation of sampling sites and
validation of methods for microbiological sampling of the environment.

Copyright © 2003 Marcel Dekker, Inc.


2. Establishment of Sampling Plans and Sites
During initial start-up or commissioning of a clean room or other controlled
environment, specific locations for air and surface sampling should be deter-
mined.
1. Consideration should be given to the proximity to the product and
whether or not the air and surfaces might be in contact with a product
or sensitive surfaces of container closure systems. Such areas should
be considered critical areas requiring more monitoring than non-prod-
uct-contact areas.
2. The frequency of sampling will depend on the criticality of specified
sites and the subsequent treatment received by the product after it has
been aseptically processed. Table 4 shows suggested frequencies of
sampling in decreasing order of frequency of sampling and in relation
to the criticality of the area of the controlled environment being sam-
pled. The sampling plans should be dynamic, with monitoring fre-
quencies and sample plan locations adjusted based on trending perfor-
mance. It is appropriate to increase or decrease sampling based on
this performance.

3. Sampling Method by ISO Air Cleanliness Standards


Establishment of Air Sampling Locations. Derive the minimum number
of sampling point locations from the formula
NL = √A
where
NL is the minimum number of sampling locations (rounded to a whole
number).

Table 4 Suggested Frequencies of Sampling on the Basis of Criticality of


Controlled Environment

Sampling area Frequency

Class 100 or better room Each operating shift


Supporting areas adjacent to class 100 Each operating shift
Other support areas (class 100,000) Twice/week
Potential product/container contact areas Twice/week
Other support areas to aseptic processing
Areas but nonproduct contact (Class 100,000 or lower) Once/week

Source: Ref. 13.

Copyright © 2003 Marcel Dekker, Inc.


A is the area of the clean room of clean air controlled space in m2. In
the case of unidirectional perpendicular airflow, the area A may be
considered as the cross section of air horizontal to the airflow.

It should be ensured that the sampling locations are evenly distributed


throughout the area of the clean room or clean zone and positioned at the height
of the work activity.

4. Establishment of Single Sample Volume Per Location


Sample a sufficient volume of air at each location that a minimum of 20 parti-
cles would be detected if the particle concentration for the relevant class were
at the class limit for the largest considered particle size.
The single sample volume VS per location is determined by using the
formula

20
VS = × 1000
Cn,m

where

VS is the minimum single sample volume per location, expressed in


liters.
Cn,m is the class limit (number of particles/m3) for the largest considered
particle size specified for the relevant class.
20 is the defined number of particles that could be counted if the parti-
cle concentration were at the class limit.

When VS is very large, the time required for sampling can be substantial. By
using the sequential sampling procedure both the required sample volume and
the time required to obtain samples may be reduced.
The sampling probe shall be positioned pointing into the airflow. If the
direction of the airflow being sampled is not controlled or predictable (e.g.,
nonunidirectional airflow), the inlet of the sampling probe shall be directed ver-
tically upward. At a minimum, sample the above-determined volume of air at
each sampling location.

5. Interpretation of Results by ISO Air Cleanliness Standard


The clean room or clean zone is deemed to have met the specified air cleanliness
classification if the averages of the particle concentrations measured at each of
the locations and, when applicable, the 95% upper confidence limit, do not
exceed the concentration limits required [13,15,19].

Copyright © 2003 Marcel Dekker, Inc.


J. Continuous Automatic Monitoring of Air
Continuous automatic air monitoring for multipoints can provide much more
information about the environment. Using the statistical analysis of the data
obtained by the continuous multipoints monitoring is the best method to monitor
the air cleanliness and to take necessary actions before the data exceed an alert
level or an action level. The method has many advantages over the data obtained
by discrete monitoring methods.
In the continuous automatic monitoring of the air quality, in which a remote
probe is used, it must be determined that the extra tubing does not have an adverse
effect on the viable airborne count. This effect should either be eliminated, or if
this is not possible, a correction factor should be introduced in reporting the re-
sults. The number of sampling ports should be calculated according to the formula
described previously, and sampling ports should be located as mentioned above.
In addition to the specified number of sampling ports, sampling ports should be
placed at the critical positions by considering the nature of the operation.
By applying this kind of continuous monitoring system, we can always
know the real-time state of air cleanliness and its trend [12]. This also affords
information as to the state of integrity of the HEPA filter without waiting for
the result of a DOP integrity test (usually performed every 6 months). A sche-
matic drawing of a continuous automatic air sampler is shown in Figure 8. An
example of monitoring data is shown in Figure 9.

K. Establishment of Microbiological Alert and Action


Levels in Controlled Environments
The principles and concepts of statistical process control are useful in establish-
ing alert and action levels and in reacting to trends. An alert level in microbio-
logical environmental monitoring is that level of micro-organism that shows a
potential drift from normal operating conditions. Exceeding the alert level is not
necessarily grounds for definitive corrective action, but it should at least prompt
a documented follow-up investigation that could include sampling plan modifi-
cations. An action level in microbiological environmental monitoring is the level
of micro-organism that when exceeded requires immediate follow-up and, if
necessary, corrective action.
Initially alert levels are established based upon the result of PQ, and re-
viewed based on the historical information gained from the routine operation of
the process in a specific controlled environment.
Trends that show a deterioration in environmental quality require attention
in determining the assignable cause and in instituting a corrective action plan
to bring the conditions back to the expected ranges. An investigation should be
implemented, however, and the potential impact should be evaluated. Although
there is no direct relationship established between the 209E or ISO air cleanliness
standard controlled environment classes and microbiological levels, the pharma-

Copyright © 2003 Marcel Dekker, Inc.


Figure 8 Schematic drawing of continuous automatic air monitoring system.

ceutical industry has been using microbial levels corresponding to air cleanliness
classes for a number of years, and these levels (shown in Table 5) have been
specified in various official compendia for evaluation of current GMP compliance
[13–16,19].

L. Methodology and Instrumentation for Quantitation of


Viable Airborne Micro-Organisms
It is generally accepted that airborne micro-organisms in controlled environ-
ments can influence the microbiological quality of the intermediate or final
products manufactured in these areas. Also, it is generally accepted that estima-
tion of the airborne micro-organisms can be affected by instruments and proce-
dures used to perform these assays.
The most commonly used samplers in the pharmaceutical and medical
device industry are impaction and centrifugal samplers. The selection, appropri-
ateness, and adequacy of using any particular sampler is the responsibility of
the user.

Copyright © 2003 Marcel Dekker, Inc.


Figure 9 Output example of continuous airborne particle measurement system.

1. Slit-to-agar air sampler (STA). This sampler is the instrument upon


which the microbial guidelines given in Table 3 for the various con-
trolled environments are based. The unit is powered by an attached
source of controllable vacuum. The air intake is obtained through a
standardized slit below which is placed a slowly revolving petri dish
containing a nutrient agar. Particles in the air that have sufficient mass
impact on the agar surface and viable organisms are allowed to grow
out. A remote air intake is often used to minimize disturbance of the
laminar flow field.
2. Sieve impactor. This apparatus consists of a container designed to
accommodate a petri dish containing a nutrient agar. The cover of the
unit is perforated, with the perforations of a predetermined size. A
vacuum pump draws a known volume of air through the cover, and
the particles in the air containing micro-organisms impact on the agar
medium in the petri dish. Some samplers are available with a cas-
caded series of containers containing perforations of decreasing size.

Copyright © 2003 Marcel Dekker, Inc.


Table 5 Comparison of Numbers of Viable Organisms Allowed by EU GMP Directive and USP Chapter <1116>

Settle plates, (cfu per


Class Air (CFU per m3) Surfaces (dfu per contact plate) 4 hr; 90 mm)

EU USP USP
(grade) ISO air class customary EU USP EU (55 m) (24–30 cm2) EU USP Descriptive

A ISO class 5 100 M 3.5 <1 3 <1 3 <1 — Criticala–c


B ISO class 5 100 M 3.5 10 — 5 — 5 —
C ISO class 7 10,000 M 100 20 25 5 (floor:10) 50 — Clean
5.5
D ISO class 8 100,000 M 200 100 50 — 50 — Controlledc, non-
6.5 sterile, support
area
a
Ref. 13.
b
Ref. 14.
c
Aseptic Processing of Health Care Product—Part 1, General—ISO 13408-1, International Organization for Standardization, Geneva (1998).

Copyright © 2003 Marcel Dekker, Inc.


These units allow for the determination of the distribution of the size
ranges of particles containing viable micro-organisms, based on which
size perforations admit the particles onto the agar plates.
3. Centrifugal sampler. The unit consists of a propeller or turbine that
pulls a known volume of air into the unit and then propels the air
outward to impact on a tangentially placed nutrient agar strip set on
a flexible plastic base.
4. Surface air system sampler. This integrated unit consists of an entry
section that accommodates an agar contact plate. Immediately behind
the contact plate is a motor and turbine that pull air through the unit’s
perforated cover over the agar contact plate and beyond the motor,
where it is exhausted. Multiple mounted assemblies are also available.
5. Gelatin filter sampler. The unit consists of a vacuum pump with an
extension hose terminating in a filter holder that can be located re-
motely in the critical space. The filter consists of random fibers of
gelatin capable of retaining airborne micro-organisms. After a speci-
fied exposure time, the filter is aseptically removed and dissolved in
an appropriate diluent and then plated on an appropriate agar medium
to establish its microbial content.
6. Settling plates. This method is still widely used as a simple and
inexpensive way to quantitatively assess the environments over pro-
longed exposure times. The exposure of open agar-filled petri dishes
or settling plates are not to be used for quantitative estimations of the
microbial contamination levels of critical environments.

One of the major limitations of mechanical air samplers is the limitation


in sample size of the air being sampled. Where the microbial level in the air of
a controlled environment is expected to contain not more than 3 cfu per cubic
meter, several cubic meters of air should be tested if the results are to be as-
signed a reasonable level of precision and accuracy. Often this is not practical.
For example, slit-to-agar samplers have an 80-L-per-min sampling capacity. If
1 cubic meter of air is tested, then it would require an exposure time of 15 min.
It may be necessary to use sampling times in excess of 15 min to obtain a
representative environmental sample. Although there are samplers capable of
very high sampling volume rates, consideration in these situations should be
given to the potential for disruption of the airflow patterns in any critical area
or to the creation of a turbulence that could increase the probability of contami-
nation. For centrifugal air samplers, a number of earlier studies showed that the
samples demonstrated a selectivity for larger particles. The use of this type of
sampler may have resulted in higher airborne counts than the other types of air
samplers because of the inherent selectivity. When selecting a centrifugal sam-
pler, the effect of the sampler on the linearity of the airflow in the controlled
zone where it is placed for sampling should be taken into consideration [13].

Copyright © 2003 Marcel Dekker, Inc.


M. Operational Evaluation of the Microbiological Status of
Aseptically Filled Products in Clean Rooms and Other
Controlled Environments
The controlled environment is monitored according to an appropriate environ-
mental monitoring program. Additional information on the evaluation of micro-
biological status can be obtained by the use of media fills. Medial fills should
be considered as a method of simulating process operations by using media,
however. Therefore, the method shows not only the environmental conditions
but also operation conditions, such as the operators’ trained level, the belt speed,
and the size (opening) of vials, which have a closer relationship with the results
of media fill test. In addition, attention has to be paid to the fact that the method
is less sensitive than other monitoring methods and can only detect contaminated
products to a level of 0.1% of falling microbes. Most of the contamination detected
by media fills are caused by process troubles such as intervention of personnel or
mechanical accident rather than the environment status or air cleanliness. The
media fill test is therefore appropriate for the evaluation of overall operations, but
not appropriate to evaluate the environment or air cleanliness [12,13].

N. An Application of Technologies for Localization of


Aseptic Processing
It is easily understood that if the aseptic operation is performed in a separated
small space from which personnel have been completely excluded, the necessity
for room classification based on particulate and environmental microbiological
monitoring requirements may be significantly reduced. In other words, critical
operations in an aseptic area should be performed in the smallest space, and
intervention by personnel should be minimized by indirect means through the
use of protective glove ports and/or half suits. Application of these methods can
minimize the chance of contamination. Following are such systems currently in
place to reduce the contamination rate in aseptic processing.

1. Barriers
In the context of aseptic processing systems, a barrier is a device that restricts
contact between operators and the aseptic field enclosed within the barrier. Bar-
riers may not be sterilized and do not always have transfer systems that allow
the passage of materials into or out of the system without exposure to the sur-
rounding environment. Barriers range from plastic curtains around the critical
production zones to rigid enclosures found on modern aseptic-filling equipment.
Barriers may also incorporate such elements as glove ports, half suits, and rapid-
transfer ports.

Copyright © 2003 Marcel Dekker, Inc.


2. Isolator
This technology is used for a dual purpose. One is to protect the product from
contamination from the environment and/or personnel during filling and closing
operation by keeping the air pressure inside the isolator positive, and the other
is to protect personnel or other products from deleterious or toxic products that
are being manufactured by keeping the air pressure inside the isolator negative.
Isolator technology is based on the principle of placing previously steri-
lized components (containers/products/closures) into a sterile environment.
These components remain sterile during the whole processing operation, since
no personnel or nonsterile components are brought into the isolator. The isolator
barrier is an absolute barrier that does not allow for interchanges between the
protected and unprotected environments. Isolators either may be physically
sealed against the entry of external contamination or may be effectively sealed
by the application of continuous overpressure. Manipulations of materials by
personnel are done via the use of gloves, half suits, or full suits. All air entering
the isolator passes through either a HEPA or an UPLA filter, and exhaust air
typically exits through a HEPA-grade filter. Per-acetic acid and/or hydrogen
peroxide vapor are commonly used for the surface sterilization of the isolator
unit’s internal environment. Since barrier systems are designed to reduce human
intervention to a minimum, remote sampling systems should be used in lieu of
personnel intervention. In general, once the validation establishes the effective-
ness of the barrier system, the frequency of sampling to monitor the microbio-
logical status of the aseptic processing area can be reduced, as compared to the
frequency of sampling of classic aseptic processing systems.
Continuous total particulate monitoring can also provide assurance that
the air filtration system within the isolator is working properly, just as in the
normal environmentally controlled area [13].

3. Summary of Air Handling Systems Validation


1. Determination of required air quality
2. Design of total air treatment system
3. Supply of air to the room
a. Amount of air
b. Locations of air supply
c. Air velocity
d. Airflow pattern
e. Exchange ratio
f. Return ratio
g. Temperature and humidity
h. Amount of exhaust
i. Location of exhaust
j. Pressure differential among the rooms

Copyright © 2003 Marcel Dekker, Inc.


4. Qualification of air cleanliness
a. Frequency of air monitoring
b. Location of sampling
c. Method of evaluation
5. Qualification of design, installation, operation, and performance, in-
cluding monitoring
6. Monitoring of air quality; monitoring data should be evaluated by
comparing with the protocol and summarized as a validation docu-
ment
7. Corrective actions, if necessary
8. Change control
9. Maintenance

V. THE VALIDATION REPORT

Validation reports are written at the conclusion of the equipment IQ and OQ


and when process validation is completed. The reports should be stand-alone
documents containing all pertinent information because they will serve as pri-
mary documentation for later FDA regulatory inspections and as reference docu-
ments when changes to the system are planned and the need for revalidation is
under consideration.
Like the validation protocol, the validation report has a standard format.
It begins with a brief executive summary, in which the major findings and rec-
ommendations are presented. All protocol deviations are identified here, along
with a brief explanation of the reason for the deviation and its impact, if any,
on the outcome of the validation. Next is a discussion section, in which all
findings, conclusions, and recommendations noted in the executive summary are
explained in detail. Topics should be presented in the order in which they appear
in the protocol. Protocol deviations are fully explained and justified, and a judg-
ment is made by a competent individual (or individuals) regarding their impact on
the validation study. Data tables and attachments should be referenced as needed.
Conclusions and recommendations is the next section. Here, a statement
is made regarding the validation status of the water or air treatment system and
the possible need for additional validation studies focusing on some aspect or
component of the system.
The last section of the report is a list of attachments. Because the report
will be the official, complete file on the water or air treatment system validation,
it must contain raw data, drawings, manuals, tables, instrument calibration re-
ports, and a copy of the validation protocol along with any protocol addenda.
The report is then endorsed and dated by designated representatives of each unit
involved in the water or air treatment system validation.

Copyright © 2003 Marcel Dekker, Inc.


REFERENCES

1. ICH. Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients.


Q7A (March 15, 2000).
2. General information (1231): Water for pharmaceutical purposes. U.S. Pharmacopeia.
vol. 25. Rockville, MD: U.S. Pharmacopeial Convention, pp. 2261–2270 (2002).
3. Water for injection, purified, sterile. U.S. Pharmacopeia. vol. 25. Rockville, MD:
pp. 1809–1890 (2002).
4. Purified water, water for injection. European Pharmacopeia. 3rd ed. Strasbourg:
European Pharmacopeia Convention, pp. 1723–1724 (1996).
5. Water for injection. Japanese Pharmacopeia. 14th ed. Tokyo: Hirokawa, p. D-751
(2001).
6. Meltzer, T. H. Pharmaceutical Water Systems. Littleton, CO: Tall Oaks, p. 683–
691 (1996).
7. FDA. FDA’s proposed current good manufacturing practices (GMP) for regs. for
large volume parenterals (LVP). Fed Reg (June 1, 1976). Preliminary Concept Paper
of Sterile Drug Products Produced by Aseptic Processing, draft paper, Sept. 27, 2002.
8. Carton, F. J., Agalloco, J. P., Artiss, D. H. Validation of Aseptic Pharmaceutical
Process: Water System Validation. New York: Marcel Dekker (1996).
9. Pharmaceutical Engineering Guide Vol. 4: Water and Steam Guide. Tampa, FL:
ISPE, (1997).
10. Code of Federal Regulation Title 21, Part 211. Current good manufacturing practice
for finished pharmaceuticals (2002).
11. Cleaning and Cleaning Validation: A Biotechnology Perspective. Bethesda, MD:
PDA (1996).
12. Kawamura, K. Scale-up, validation and manufacturing of microspheres in the “sus-
tained release injectable products.” J. Senior and M. Radomsky, eds. Englewood,
CO: InterPharm Press (2000).
13. General information <1116>: Microbiological evaluation of clean rooms and other
controlled environments. U.S. Pharmacopeia. vol. 25. Rockville, MD: U.S. Pharma-
copeial Convention, p. 2206–2212 (2002).
14. EU. GMP annex: Manufacturing of sterile products (1996).
15. Federal Standard 209E. Airborne Particulate Cleanliness Classes in Cleanroom and
Clean Zone (Sept. 11, 1992).
16. Japanese Pharmacopeia. 14th ed. suppl. Tokyo: Hirokawa (2001).
17. Pharmaceutical Engineering Guide Vol. 3: Sterile Manufacturing Facilities.
Tampa, FL: ISPE, (1997).
18. FDA/ISPE. Pharmaceutical Engineering Guide. vol. 1. Tampa, FL.
19. Classification of Airborne Particulates, in Cleanrooms and Associated Controlled
Environments—Part 1, ISO 14644-1, Geneva: International Organization for Stan-
dardization (1999).
20. WHO GMP. Good Practices in Manufacturing of Pharmaceutical Products in
WHO Expert Committee on Specifications for Pharmaceutical Preparations, 32
edition, Geneva (1992).

Copyright © 2003 Marcel Dekker, Inc.